1. Field of the Invention
The present invention is directed to a vaccine and method of making a vaccine for Isospora suis based upon recombinantly derived sporozoite protective antigens, recombinantly derived merozoite protective antigens, and/or cell cultured derived merozoites.
2. Description of the Background Art
Isospora suis and Swine Coccidiosis
Coccidiosis was first reported in swine as early as 1878. However, the causative agent of this disease was not identified as Isospora suis until 1934. Isospora suis cost swine producers in the U.S. an estimated $101,000,000 from July, 1992 to May, 1993. This amounts to a loss of more than $1 billion to swine producers in the U.S. over the last ten years, assuming that the infection rates have not varied significantly. There is no current vaccine on the market, no coccidiostats are licensed for swine use, non-licensed coccidiostats have not been effective, and there has been no reported method to produce the complete life cycle of this organism outside the pig.
Isospora are, with few exceptions, parasites of the gastrointestinal tract. Isospora, as members of the genus Eimeriidae, are usually monoxenous, i.e., use only one host, although some species of Isospora have been shown to use transport hosts.
Neonatal porcine coccidiosis is caused by Isospora suis. Naturally infected piglets develop diarrhea 5-10 days after birth, become dehydrated, and lose weight. Morbidity is high and mortality is usually low to moderate. Lesions of villous atrophy, villous erosion, and fibrinonectroic enteritis have been described from experimentally and naturally infected piglets. The majority of clinical cases of Isospora suis are observed in pre-weaned pigs, with about one third of the cases occurring in post-weaned pigs. Infections are less severe in older pigs than in piglets.
Isospora are parasites of the intestinal tract and have an obligate intracellular merogony and gametogony, single host life cycle, extra-intestinal sporogony, and oocysts with two sporocysts, each of which contains four sporozoites.
Upon ingestion of the sporulated oocysts, the motile infective stages, the sporozoite, are released and enter a host cell. The sporozoites differentiate into meronts and initiate the first multiplication cycle (merogony), which is asexual. Nuclear division and growth produce multiple merozoites from a meront, the number of which depends on the species of the organism. The meront ruptures the cell and the released motile merozoites invade new host cells to initiate a second generation of meronts. In Isospora suis, Type-1 meronts are binucleate and divide to form two merozoites, while type-2 meronts are multinucleate and divide to form many type-2 merozoites. Merogony continues for several generations, usually two to three. Eventually the released merozoites produce the sexual stages. Some merozoites become microgamonts in which multiple nuclear divisions take place and many microgametes are formed. Other merozoites form macrogametes, which have one nucleus that does not divide. A macrogamate is fertilized by one microgamete to form a zygote. A wall of one or more layers develops around the zygote to form an oocyst, which is released into the lumen of the host's gut and excreted in the feces. Outside the body the oocyst sporulates into the infective stage. Once the oocysts are excreted from an infected animal, reduction division comes into play. The sporulation of the oocysts takes place after the oocysts have been shed from the animal. Sporulation is dependent upon temperature. The first observable nuclear division occurs at the time the sporont divides to form two spherical sporoblasts. Nuclear division occurs again and the nuclei migrate to the poles of the developing sporocysts. As a result of a final nuclear division, four sporozoites are produced in each sporocysts.
Excystation occurs in the newly infected host and is the process by which sporozoites are released from sporocysts and eventually from the oocysts. Excystation of oocysts will occur in almost any animal, but the released sporozoites will fail to produce infection in any but the normal hosts. According to several workers, excystation is a two-step process. First, the oocyst wall must be altered to make it permeable to bile and pancreatic enzymes. This is thought to be accomplished by stomach acids and in the reducing environment of the stomach. The bile and pancreatic enzymes in the intestine are thought to cause activation of sporozoites and dissolution of the Stieda body. The sporozoites leave the sporocysts through the opening left by the disintegration of the Stieda body and leave the oocysts through breaks or gaps that develop in the oocysts wall. Isospora that lack a Stieda body excyst by collapse of the sporocyst wall along plate-like junctions. The collapse releases the sporozoites into the oocysts, from which they then escape.
The Vertebrate Immune System
The ability of vertebrates to protect themselves against infectious microbes, toxins, viruses, or other foreign macromolecules is referred to as immunity. The art distinguishes between natural, and acquired or specific immunity. Natural immunity is comprised of defense mechanisms which are active before exposure to microbes or foreign macromolecules, are not enhanced by such exposure, and do not distinguish among most substances foreign to the body. Acquired or specific immunity comprises defense mechanisms which are induced or stimulated by exposure to foreign substances.
In vertebrates, the mechanisms of natural and specific immunity cooperate within a system of host defenses, the immune system, to eliminate foreign invaders. The events by which the mechanisms of specific immunity become engaged in the defense against invading microorganisms cancer cells, etc. are termed immune responses. Vertebrates have two basic immune responses: humoral and cellular. Humoral immunity is provided by B lymphocytes, which, after proliferation and differentiation, produce antibodies which circulate in the blood and lymphatic fluid. Cellular immunity is provided by the T cells of the lymphatic system. The cellular immune response is particularly effective against fungi, parasites, intracellular viral infections, cancer cells and foreign matter, whereas the humoral response primarily defends against the extracellular phases of bacterial and viral infections.
An "antigen" is a foreign substance which is recognized (specifically bound) by an antibody or a T-cell receptor, regardless of whether it can induce an immune response. Foreign substances inducing specific immunity are termed "immunizing antigens", or "immunogens". An "hapten" is an antigen which cannot, by itself, elicit an immune response (though a conjugate of several molecules of the hapten, or of the hapten to a macromolecular carrier, might do so).
The immune system has evolved so that it is able to recognize surface features of macromolecules that are not normal constituents of the host. A foreign molecule which is recognized by the immune system (e.g., bound by antibodies), regardless of whether it can itself elicit is called an "antigen", and the portion of the antigen to which an antibody binds is called the "antigenic determinant", or "epitope". When the antigen is a polypeptide, it is customary to classify epitopes as being linear (i.e., composed of a contiguous sequence of amino acids along the polypeptide chain) or nonlinear (i.e., composed of amino acids brought into proximity as a result of the folding of the polypeptide chain). (The nonlinear epitopes are also called "conformational" because they arise through the folding of the polypeptide chain into a particular conformation.)
To cope with the immense variety of epitopes encountered, the immune system of a mammalian individual contains an extremely large repertoire of lymphocytes. Each lymphocyte clone of the repertoire contains surface receptors specific for one epitope. It is estimated that the mammalian immune system can distinguish at least 10.sup.8 distinct antigenic determinants.
An initial or primary immune response to a foreign antigen enhances the ability of the immune system to respond again to that antigen (in a secondary immune response). This feature of specific immunity is called immunologic memory. Secondary immune responses are often more effective than primary responses.
Lymphocytes are the agents of antigenic specificity in the immune response. They can be divided into two groups. One group, the "B-lymphocytes" or "B-cells", play a central role in the production of antibodies. Antibodies (immunoglobulins, Ig's) are proteins capable of binding antigens, and exerting effector functions that are involved in the elimination of foreign antigens. The other group consists of T-lymphocytes or T-cells that perform a variety of functions including help for B-cells, production of delayed-type hypersensitivity reactions, and specific killing of virus-infected cells.
Normally, immune responses progress toward effector mechanisms characteristic of both B and T-lymphocytes. However, in the course of most immune responses, either B or T lymphocytes assume a dominant role, with less substantial participation of the respective other type of lymphocyte. Immune responses whose effector mechanisms are mediated preponderantly through B-cells and antibodies are termed humoral immune responses. Those responses wherein T-cells mediate the more important effector functions are referred to as cell-mediated or cellular immune responses.
B-cells constitute the population of lymphocytes central to humoral immune responses. Each clone of B-lymphocytes expresses membrane immunoglobulins (membrane Ig's, surface-bound antibody molecules) that function as antigen receptors with one unique epitope specifically per. B-lymphocyte clone. These membrane Ig molecules (antigen receptors) are the sole source of B-cell specificity. Antigens that contain an epitope complementary to the membrane Ig will bind to the antigen receptor. Such antigens are also referred to as cognate antigens of the antibody. On protein antigens, antibodies can bind linear determinants (epitopes formed by adjacent amino acid residues in the covalent sequence), or conformational determinants, which are formed by amino acid residues from separate portions of the linear polypeptide that are specially juxtaposed by polypeptide folding. Binding to the antigen receptor (membrane Ig) will result in differentiation and clonal proliferation of the B-lymphocyte. Some of its progeny will differentiate into mature plasma cells which are specialized in the synthesis of antibodies corresponding in epitope specificity to the membrane Ig by which the B-lymphocyte had initially bound the antigen.
By an effector mechanism typical of humoral immune responses, antibodies will bind to cognate epitopes on the surface of invading target cells, e.g., bacteria. Following antibody binding, the components of the complement system will sequentially attach to the target cell-antibody complex, resulting ultimately in the rupture of the target cell membrane and killing of the target cell. By another antibody-mediated effector mechanism, target antigens are bound and cross-linked (opsonized) by antibodies, and are thus prepared for ingestion and subsequent destruction by phagocytes of reticuloendothelial origin, such as granulocytes or macrophages.
The antibody itself is an oligomeric molecule, classified, according to its structure, into a class (e.g., IgG) and subclass (e.g., IgGl). IgG molecules are the most important component of the humoral immune response and are composed of two heavy (long) and two light (short) chains, joined by disulfide bonds into a "Y" configuration. The molecule has both variable regions (at the arms of the "Y") and a constant region (the hinge and base of the "Y"). The regions are so named because antibodies of a particular subclass, produced by a particular individual in response to different antigens, will differ in the variable region but not in the constant region. The variable regions themselves are composed of both a relatively invariant framework, and of hypervariable loops, which confer on the antibody its specificity for a particular epitope. An antibody binds to an epitope of an antigen as a result of molecular complementarity. The portions of the antibody which participate directly in the interaction is called the "antigen binding site", or "paratope". The antigens bound by a particular antibody are called its "cognate antigens".
Surface IgM is the first antibody to appear on the surface of B cells, and secreted IgM is the major component of the primary immune response. The affinity of IgM antibodies is relatively low, but this is offset by their multivalency. They are particularly effective against polyvalent antigens. IgM antibodies are very effective in inhibiting pathogens by agglutination (via complement fixation).
IgG antibodies are the major component of the secondary immune response to T dependent antigens. Certain subclasses of IgG antibodies can activate complement. Some subclasses are transferred, by specific receptors, across the placenta. IgG antibodies can also sensitize targets to eosinophils.
IgA antibodies are the major immunoglobulin component of secretions, and are transported across epithelia, aiding the body's exterior defenses.
IgE antibodies link the immune system to inflammatory effectors.
An antibody of one animal will be seen as a foreign antigen by the immune system of another animal, and will therefore elicit an immune response. Some of the resulting antibodies will be specific for the unique epitopes (idiotype) of the variable region of the immunizing antibody, and are therefore termed anti-idiotypic antibodies. These often have immunological characteristics similar to those of an antigen cognate to the immunizing antibody. Anti-isotypic antibodies, on the other hand, bind epitopes in the constant region of the immunizing antigen.
The typical effector phase of cell-mediated or cellular immune responses involves lysis or killing of target cells by cytotoxic or cytolytic T-lymphocytes (CTLs) through direct cell-to-cell contact. Molecules from two diverse families of cell-surface glycoproteins, the T-cell receptors (TCRs) and the major histocompatibility complex (MHC) type I glycoproteins, are the key elements of specificity in the CTL response to foreign antigens. T-cell receptors (TCRs) recognize short, linear peptide determinants of 8-24 amino acids, the generation of which usually requires unfolding and proteolytic fragmentation ("processing") of the antigenic protein. They can also recognize oligosaccharide determinants. Unlike antibodies, T-cell receptors cannot recognize conformational epitopes.
The second difference in antigen recognition by antibodies and T-cell receptors is the involvement of a third molecule that performs the role of presenting the antigen to the T-cell receptor. For B-cells, such molecules are not necessary, as the membrane Ig (antibody) forms a stable bimolecular complex with the antigenic protein. For T-cells, the antigenic peptide must be bound by an MHC glyco-protein, and it is this complex of MHC molecule plus peptide that forms the structure recognized by the T-cell receptor. MHC glycoproteins are thus peptide-binding proteins which function as antigen-presenting molecules.
Poultry Coccidiosis (Eimeria) Vaccines
While the art has not developed a vaccine against Isospora suis, the cause of coccidiosis in pigs, the poultry coccidiosis agents, which are various species of the genus Eimeria, have proven to be more tractable.
Eimeria necatrix has been attenuated by inoculating sporozoites into embryonated eggs, passaging 20-60 times, and then harvesting oocysts. An oocyst suspension can then be used as a live vaccine against coccidiosis in poultry. See Shirley, U.S. Pat. No. 4,438,097.
Sporozoites have been isolated from oocysts and used directly as vaccines. Bhogal, et al. U.S. Pat. No. 5,068,104, teaches keeping Eimeria sporozoites alive, after emergence, by encapsulating them. The microcapsulated sporozoites are administered to baby chicks.
Murray, EP 167,443 (Merch & Co.) and U.S. Pat. No. 4,639,372 suggests that extracts from sporozoites or sporulated oocysts of Eimeria tenella, which do not contain viable or intact parasites, can be used to protect chickens from coccidiosis. According to Murray, these extracts "contain at least 15 polypeptides, many of which are associated with the surface of the sporozoite and induce good immune responses." In Murray, U.S. Pat. No. 4,724,145, similar use is made of an E. acervulina extract to protect against E. acervulina, E. tenella, and E. maxima.
There has been considerable interest in producing particular Eimeria sporozoite antigens by recombinant DNA techniques, as summarized below:
______________________________________ 25 kDa E. acervulina antigen Jacobson U.S. Pat. No. ac-1b; 21.6 kDa E. accervulina 5,273,901 and antigenic fragment ac-6b; 16.6 WO92/04460 kDa E. tenella antigen tc-7a; 3.5 kDa E. tenella antigenic fragment tc-89; 7.8 kDa E. tenella antigenic fragment tc-10a, all immunoreactive with anti-sporozoite monoclonal antibodies 25 kDa E. tenella antigen Anderson U.S. Pat. No. 5,279,960 and WO90/00403 25 kDa E. tenella antigen composed Andrews, U.S. Pat. No. of two disulfide bonded polypeptides 4,874,705 and (12 and 8 kDa). Newman EP 231,537 (Solvay) 50-65 kDa E. maxima antigen Harwood, WO92/16627 (Campbell Soup) 100 kDa E. acervulina antigen Kok, EP 519,547 (Akzo) ______________________________________
Kok also identified 20, 45, 100 and 200 kDa E. acervulina merozoite antigens. Schenkel, U.S. Pat. No. 4,650,676 discussed 300.+-.50, 130.+-.20 and 18.+-.3 kDa merozoite antigens.
Comparison of Isospora and Eimeria
Isospora and Eimeria are genera which contain parasites which are similar in many respects but different in other, significant respects. Both cause coccidiosis in warm-blooded animals, and, in general, the route of infection, the general reproductive cycle, and clinical diseases caused by Isospora and Eimeria are also similar. However, Isospora suis is only known to reproduce itself in swine and is the only pathogen to cause coccidiosis in pigs, while Eimeria species have been observed in a wide variety of animals while not necessarily causing disease in these animals. Eimeria species have been observed in swine while not necessarily causing disease in swine, and can cause disease in avians, including chickens and turkeys.
Because of this difference in hosts, the immune response to an Isospora suis infection differs from that to an infection caused by Eimeria. The immune response of birds is quite different from that of pigs in many respects, including mechanisms of preventing or limiting coccidial infections.
Birds have a bursa where B-cells are known to differentiate, and pigs do not have a bursa. The bursa equivalent in swine for the mucosal system is understood to be in the intestines (American Society of Biochemist and Molecular Biologist convention in San Francisco, Calif., in January, 1989). The digestive and reproductive systems are also very different. These differences inhibit the direct application of chicken Eimeria research to swine Isospora suis vaccine development.
The antigens presented by the two genera are also different. The immunodominant Eimeria acervulina sporozoite antigen (previously described as p160/p240) is a 19 kilodalton antigen present in several Eimeria species (cf. Laurent et al. in Mol. Biochem. Parasit. 63: 79-86, 1994), while that of Isospora suis is 207 and 218 kilodaltons. Eimeria bovis have merozoite surface proteins with molecular weights between 15 and 18 kD, and have sporozoite surface proteins of 28, 77 and 183 kD (cf. Reduker et al., J. Parasit. 72(6): 901-907, 1986), while Isospora suis does not.
Isospora species are in general more difficult to work with than Eimeria species for the following reasons:
(a) fewer oocysts are produced;
(b) more fat is present in mammalian diets than in avian diets, making purification of oocysts more difficult;
(c) the oocyst wall is thinner in Isospora than Eimeria, making it more difficult to sterilize and store;
(d) mammalian hosts not previously exposed to Isospora are more difficult to find and more costly to purchase than the Eimeria avian hosts (cf. Lindsay et al., Parasitology Today, 10(6): 214-220, 1994).
The conventional belief is that it is not possible to confer passive immunity to Isospora suis. Baekbo et al., Proceeding of the 13th IPVS Congress, Bangkok, Thailand, 26-30 June, 1994, state, "With the set-up in the present experimental study it was impossible to transfer a passive protective immunity against I. suis infection from sows to their offspring."
Development of Isospora Suis in Cell Culture and in Embryos
Lindsay and Current, J. Protozool., 31:152-5 (1984) reported the "complete" development of Isospora suis in chicken embryos. The allantoic cavities were inoculated with sporozoites. The authors obtained Type 1 meronts and merozoites, Type 2 meronts and merozoites, and mature microgamonts, macrogamonts, and oocysts. However, sporulation did not occur. The disadvantages of an embryo system include the following: (a) the yield of merozoites and oocysts is very low; (b) The oocysts are not viable (alive); and (c) The use of eggs for vaccine production is more labor intensive and would require safety testing for viruses that may be present in the embryos.
Fayer, et al., Proc. Helminthol. Soc. Wash., 51:154-9 (1984) described an unsuccessful effort to induce intracellular development of sporozoites inoculated into Madin-Darby bovine kidney (MDBK), embryonic bovine trachea (EBTr), bovine colon (BC) and porcine kidney (PK) cell cultures.
Lindsay and Blagburn, Veter. Parasitol., 24:301-4 (1987) inoculated primary porcine kidney (PPK) and fetal bovine kidney (PFBK) cell cultures with sporozoites of Isospora suis. Motile merozoites and binucleate Type I meronts were observed in both cultures. Multinucleate Type II meronts, which did not form merozoites, developed in PPK cell cultures only.
Lindsay and Blagburn, Parasitol. Today, 10:214 (1994) summarized the then state of the art as follows:
several mammalian Isospora species have been grown in cell cultures . . . with several divisions occurring by endodyogeny. Only I. rivolta and I. suis have produced multinucleate (more than two nuclear) schizonts in cell cultures and these schizonts did not reach maturity. Sexual stages and oocysts have not developed in cell cultures. Continuous cultivation of an Isospora species has not been achieved.
In contrast, with Eimeria, more than two decades earlier, the art had discovered how to progress from sporozoites to functional oocysts in cell culture. See Doran, Proc. Helminth. Soc., 37:84 (1970).